Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) mediates DNA replication of terminal repeat (TR) DNA to enable viral episome persistence in latently infected cells. Southern blotting is routinely used to detect LANA-replicated DNA. We developed and validated a real-time PCR assay for TR-associated DNA and compared it with Southern blot analysis. Both PCR and Southern blot detected LANA-replicated DNA, but the PCR assay was more rapid and did not require radioisotope. PCR detection at 24 and 72 hours post-transfection demonstrated rapid loss of transfected TR DNA. LANA, and to a lesser extent a moderately deficient LANA mutant, reduced the rate of DNA loss through addition of replicated TR DNA and reduction in the loss of non-replicated DNA, the latter of which is consistent with LANA’s nuclear segregation function. Therefore, this work develops a rapid, sensitive, and quantitative PCR (qPCR) assay to detect KSHV LANA-replicated DNA and demonstrates that LANA reduces TR DNA loss after transfection through replication and nuclear partitioning of TR DNA.

The latency-associated nuclear antigen of Kaposi’s sarcoma-associated herpesvirus interacts preferentially with the terminal repeats of the genome in vivo and this complex is sufficient for episomal DNA replication

Fejer, G; Medveczky, MM; Horvath, E; Lane, B; Chang, Y; Medveczky, PG

Latency-associated nuclear antigen (LANA) cooperatively binds to two sites within the terminal repeat, and both sites contribute to the ability of LANA to suppress transcription and to facilitate DNA replication